STIMULATION OF THE ADENOVIRUS MAJOR LATE PROMOTER IN-VITRO BY TRANSCRIPTION FACTOR USF IS ENHANCED BY THE ADENOVIRUS DNA-BINDING PROTEIN

Previous studies have shown that the sequence-independent adenovirus D NA binding protein (DBP) increases transcription from several promoter s, notably from the adenovirus major late promoter (MLP) and the adeno -associated virus P5 promoter, both of which contain a USF/MLTF bindin g site. In order to study this mechanism, we have investigated the eff ects of DBP on the binding of USF/MLTF to MLP and on transcription fro m MLP by a reconstituted in vitro system. As shown by gel retardation and DNase I footprinting, upon saturation of DNA, DBP enhances the bin ding affinity of USF43 to the promoter three- to fourfold without chan ging the footprint pattern. In contrast, the binding of the TATA box b inding protein to the promoter is not influenced by DBP. No protein-pr otein interactions between DBP and USF43 could be observed in the abse nce of DNA, suggesting that enhanced binding is caused by a change in DNA structure induced by the DBP-DNA complex. Employing a transcriptio n system reconstituted with purified general transcription factors, we show that USF43 enhances basal transcription and that USF43-dependent transcription is further increased by DBP, while DBP alone does not h ave an effect on basal transcription. Our results suggest that transcr iption enhancement by DBP is based on a specific increase in the bindi ng of a transcription factor to a promoter through subtle changes in D NA structure, similar to the mechanism by which DBP stimulates the ini tiation of DNA replication.